Bottom Line:
The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes.The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein.In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues.

DEV105593F1: Identification of putative macrophage lineage-specific regulatory elements in the first intron of the chicken CSF1R gene. (A) mVista alignment (http://gsd.lbl.gov/vista/) of the CSF1R first intron comparing chicken (Gg) with turkey (Mg), Adélie penguin (Pa), zebrafinch (Tg), rifleman (Ac), ostrich (Sc) and Chinese softshell turtle (Pc). Conserved regions (>70% homology over 100 bp window) are shaded. The positions of four major conserved non-coding elements (CNEs) are boxed and numbered. (B) Pustell DNA matrix alignment of the avian/reptile CSF1R CNE2 and CNE3. The unbroken diagonal lines represent regions of high sequence conservation, and the broken and offset lines indicate that an insertion has occurred in the chicken/turkey lineage in comparison with the other species shown here. The avian-specific CNE2 is highlighted in red; the CNE-3, which is conserved in birds and turtle sequence, is highlighted in blue. (C) Alignment of mammalian Fms-intronic regulatory element (FIRE) with the CSF1R CNE3 region in birds/reptiles. Species sequences from top to bottom are human, mouse, platypus, turtle, alligator, Adélie penguin, budgerigar, ostrich, rifleman, zebrafinch, duck, turkey, chicken and consensus sequence. Arrows indicate the location of the two murine FIRE transcription start sites (Sauter et al., 2013) and conserved transcription factor binding sites are also shown. (D) Sequence of the chicken macrophage lineage-specific regulatory element used in this study: binding sites for PU.1, C/EBP, AP1, SP1 and AML1 are identiﬁed. The avian-specific CNE2 is highlighted in red and the avian-reptile-mammal conserved CNE3 is in blue.

Mentions:
Conservation of sequences within the first intron of avian CSF1R genes was evident from an alignment of chicken and zebrafinch CSF1R genomic sequences (Garceau et al., 2010). The availability of many more genome sequences has enabled us to align sequences of four additional bird species and a reptile with chicken, to identify potential regulatory sequences in the chicken by their conservation between distantly related species. The first intron of CSFIR contains four conserved non-coding elements (CNEs) that are present in all birds (Fig. 1A). Pustell DNA matrix alignment of CNE2 and CNE3 suggests that they were formed in the galliforme lineage by an insertion into an original single CNE (Fig. 1B). CNE3 is also conserved in turtles (Fig. 1A,B). Comparison of mammalian FIRE with CNE3 in birds and reptiles identified several regions of ultra-conserved sequence (Fig. 1C). These ultra-conserved regions contain the precise binding sites of transcription factors AP1 and PU.1 that are occupied in the macrophage nucleus (Tagoh et al., 2002) and are required for macrophage lineage-specific transcription of Csf1r in mice (Fig. 1C,D) (Sauter et al., 2013). To test the function of the candidate chicken FIRE sequence, we produced eGFP reporter constructs containing the chicken CSF1R promoter region (Garceau et al., 2010) with or without the CNE3 region (supplementary material Fig. S1A,B). eGFP expression was detected in stably transfected HD11 macrophage cells only when CNE3 was included, whereas no expression was detected in transfected DF-1 fibroblast cells (supplementary material Fig. S1C). Based upon sequence conservation and function, we refer to CNE-3 as chicken FIRE.Fig. 1.

DEV105593F1: Identification of putative macrophage lineage-specific regulatory elements in the first intron of the chicken CSF1R gene. (A) mVista alignment (http://gsd.lbl.gov/vista/) of the CSF1R first intron comparing chicken (Gg) with turkey (Mg), Adélie penguin (Pa), zebrafinch (Tg), rifleman (Ac), ostrich (Sc) and Chinese softshell turtle (Pc). Conserved regions (>70% homology over 100 bp window) are shaded. The positions of four major conserved non-coding elements (CNEs) are boxed and numbered. (B) Pustell DNA matrix alignment of the avian/reptile CSF1R CNE2 and CNE3. The unbroken diagonal lines represent regions of high sequence conservation, and the broken and offset lines indicate that an insertion has occurred in the chicken/turkey lineage in comparison with the other species shown here. The avian-specific CNE2 is highlighted in red; the CNE-3, which is conserved in birds and turtle sequence, is highlighted in blue. (C) Alignment of mammalian Fms-intronic regulatory element (FIRE) with the CSF1R CNE3 region in birds/reptiles. Species sequences from top to bottom are human, mouse, platypus, turtle, alligator, Adélie penguin, budgerigar, ostrich, rifleman, zebrafinch, duck, turkey, chicken and consensus sequence. Arrows indicate the location of the two murine FIRE transcription start sites (Sauter et al., 2013) and conserved transcription factor binding sites are also shown. (D) Sequence of the chicken macrophage lineage-specific regulatory element used in this study: binding sites for PU.1, C/EBP, AP1, SP1 and AML1 are identiﬁed. The avian-specific CNE2 is highlighted in red and the avian-reptile-mammal conserved CNE3 is in blue.

Mentions:
Conservation of sequences within the first intron of avian CSF1R genes was evident from an alignment of chicken and zebrafinch CSF1R genomic sequences (Garceau et al., 2010). The availability of many more genome sequences has enabled us to align sequences of four additional bird species and a reptile with chicken, to identify potential regulatory sequences in the chicken by their conservation between distantly related species. The first intron of CSFIR contains four conserved non-coding elements (CNEs) that are present in all birds (Fig. 1A). Pustell DNA matrix alignment of CNE2 and CNE3 suggests that they were formed in the galliforme lineage by an insertion into an original single CNE (Fig. 1B). CNE3 is also conserved in turtles (Fig. 1A,B). Comparison of mammalian FIRE with CNE3 in birds and reptiles identified several regions of ultra-conserved sequence (Fig. 1C). These ultra-conserved regions contain the precise binding sites of transcription factors AP1 and PU.1 that are occupied in the macrophage nucleus (Tagoh et al., 2002) and are required for macrophage lineage-specific transcription of Csf1r in mice (Fig. 1C,D) (Sauter et al., 2013). To test the function of the candidate chicken FIRE sequence, we produced eGFP reporter constructs containing the chicken CSF1R promoter region (Garceau et al., 2010) with or without the CNE3 region (supplementary material Fig. S1A,B). eGFP expression was detected in stably transfected HD11 macrophage cells only when CNE3 was included, whereas no expression was detected in transfected DF-1 fibroblast cells (supplementary material Fig. S1C). Based upon sequence conservation and function, we refer to CNE-3 as chicken FIRE.Fig. 1.

Bottom Line:
The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes.The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein.In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues.